Microbeads have been used as surrogate cell standards in various biological fields for many years. This is especially true since highly uniform microbeads, in the 2-15 micron diameter range, have been first produced by John Ugelstad. As a particle surrogate, polymeric microbeads have high physical stability, as well as the ability to be labeled with the same fluorochromes and dyes as used to label biological cells. Moreover, a great deal of work has been addressed to make microbeads useful as fluorescent particle reference and quantitative standards. When labeled with such fluorochromes and dyes, these microbeads exhibit many of characteristics as labeled biological cells, as determined by instrumentation of at least one of flow cytometry. Specifically, fluorochrome labeled microbeads appear to have similar forward and side light scatter properties, as well as similar spectral and intensity fluorescent properties as biological cells.
However, the fluorescence intensity distribution profile of microbeads and biological cells labeled with the same fluorochrome can appear vastly different. For example human lymphocytes labeled with a fluorescently conjugated CD8 antibody, has a fluorescence intensity distribution, as shown in FIG. 1. This distribution is really the combined response of CD8 suppressor cells, the tall intense population on the right end of the distribution, and CD8 cyto-toxic cells, the low widely spread population to the left of the CD8 suppressor cells.
Much effort in the manufacture of microbeads is focused on producing highly uniform physical properties, of at least one of size, volume and fluorescence intensity. Such populations are highly sort after for use as size and fluorescence standards in fields of at least one of flow cytometry and fluorescence microscopy. Normally, these methods of production ensure that fluorochromes or dyes are taken up by the microbeads in a highly uniform fashion resulting in a very tight fluorescence intensity distribution, as shown in FIG. 2.
However, it stands to reason that, the closer the standards mimic analyte being measured, the better the instrument performance can be evaluated to measure the analyte. An example to illustrate this may be found when counting CD8 labeled lymphocytes. If a fluorescent population of microbeads with the usual tight intensity distribution is used as the count standard, the standard is only determining the ability of the instrument to detect and count events in a narrow range of the intensity scale, as shown in FIG. 2. However, as seen in FIG. 1, the intensity distribution of CD8 labeled lymphocytes cover a wide non-uniform intensity range. Although highly uniform intensity microbeads have served the biology and medical communities as cellular surrogates and standards well for a long time, the ability of microbeads that more closely resemble the intensity distributions of biological cells would be very welcome to these communities.